Bio Focus: High performing assay using antibody-conjugated DNA nanoswitches detects proteins

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a

Create DNA nanoswitches

b Mix nanoswitches

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Gel electrophoresis and imaging

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rotein detection is critical in many biomedical and clinical applications, including disease diagnosis and food allergen detection. Wesley Wong and colleagues at Harvard Medical School and Boston Children’s Hospital recently reported in Proceedings of the National Academy of Sciences (doi:10.1073/pnas. 1708148114) that structural changes in antibody-conjugated DNA nanoswitches can provide a cheaper, faster, and more sensitive method for protein detection than the gold standard, enzyme-linked immunosorbent assay (ELISA). They also showed that in their nanoswitchlinked immunosorbent assay (NLISA), results can be read off of mobile-imaging platforms to facilitate remote detection. The motivation for their work stems from wanting to improve sensitivity, specificity, speed, and costs and to avoid the many washing steps in current ELISA assays. First author Clinton Hansen stressed that “unlike ELISA, no enzymes are required with the nanoswitch reagents which are composed solely of DNA and antibodies that can be lyophilized to enable a long shelf life under ambient or refrigerated conditions.” To create a NLISA, Hansen and coworkers had to first conjugate antibodies to short polymers of nucleic acid known as oligonucleotides, which have complementary sequences to parts of a linear DNA molecule. This was followed by coupling the antibody-oligonucleotide to linear DNA to create the nanoswitch; see (a) in the figure. The binding of the nanoswitch to their target antigen analytes causes the linear DNA to develop a looped conformation; see (b) in the figure. Next, the team made use of gel electrophoresis, a widely used technique where negatively charged DNA molecules are separated based on size and conformation in an electric field; see (b) in the figure. Linear, unbound DNA passes through a porous gel faster than analyte-bound looped DNA, allowing DNA separation. The intensities of the

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Schematic of a nanoswitch-linked immunosorbent assay. (a) DNA nanoswitches are first made with enzymatic digestion of circular DNA into linear DNA. This is followed by conjugating oligonucleotide antibodies to the digested, linear DNA. (b) Nanoswitches bind to antigen analytes in samples causing conformational changes in DNA to form looped structures. With gel electrophoresis, linear and looped DNA can be separated by size, allowing protein detection and quantification. Credit: PNAS.

separated bands can be quantified to determine the concentrations of the analytes. The limit of detection (LOD) defined as the concentration that exceeds 3 standard deviations of the background signal for the DNA nanoswitches was found to be 0.4–1.5 femto (10–15)-Molar (fM). The researchers demonstrated the utility of NLISA with well-characterized binding between biotinylated oligonucleotide and streptavidin and achieved a LOD at 9–22 fM. They then applied their technology for detection in urine and serum, which are the most common biologic